Duocarmycin adcs showing improved in vivo antitumor activity

ABSTRACT

The present invention relates to duocarmycin-containing antibody-drug conjugates (ADCs) for use in the treatment of human solid tumours and haematological malignancies expressing HER2, in particular breast cancer, gastric cancer, bladder cancer, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, colorectal cancer, head and neck squamous cell cancer or osteosarcoma, and acute lymphoblastic leukaemia. In particular, the present invention relates to duocarmycin-containing ADCs for use in the treatment of human solid tumours with HER2 IHC 2+ or 1+ and HER2 FISH negative tissue status. Advantageously, the present invention relates to duocarmycin-containing ADCs for use in the treatment of triple negative breast cancer (TNBC).

FIELD OF THE INVENTION

The present invention relates to duocarmycin-containing antibody-drugconjugates (ADCs) for use in the treatment of human solid tumours andhaematological malignancies expressing human epidermal growth factorreceptor 2 (HER2), in particular breast cancer, gastric cancer, bladdercancer, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer,colorectal cancer, head and neck squamous cell cancer, osteosarcoma, andacute lymphoblastic leukaemia.

BACKGROUND OF THE PRESENT INVENTION

Antibodies have been conjugated to a variety of cytotoxic drugs,including small molecules that alkylate or crosslink DNA (e.g.duocarmycins and calicheamicins or pyrrolo-benzodiazepine dimers,respectively), or disrupt microtubules (e.g. maytansinoids andauristatins) or bind DNA (e.g. anthracyclines). One such ADC comprisinga humanized anti-CD33 antibody conjugated to calicheamicin—Mylotarg™(gemtuzumab ozogamicin, Wyeth)—was approved in 2000 for acute myeloidleukaemia. In 2011, the US Food and Drug Administration (FDA) approvedAdcetris™ (brentuximab vedotin, Seattle Genetics), an ADC comprising achimeric antibody to CD30 conjugated to monomethyl auristatin E (MMAE)for treatment of Hodgkin's lymphoma and anaplastic large cell lymphoma.

Duocarmycins, first isolated from a culture broth of Streptomycesspecies, are members of a family of antitumor antibiotics that includeduocarmycin A, duocarmycin SA, and CC-1065. These extremely potentagents allegedly derive their biological activity from an ability tosequence-selectively alkylate DNA at the N3 position of adenine in theminor groove, which initiates a cascade of events leading to tumour celldeath.

WO2011/133039A discloses a series of novel analogues of theDNA-alkylating agent CC-1065 and HER2-targeting ADCs thereof. In Example15, a number of trastuzumab-duocarmycin conjugates were tested againstN87 (i.e. HER2 IHC (immunohistochemistry) 3+ gastric tumour) xenograftsin nude mice. The results are shown in FIGS. 4A, 4B and 4C. Aftertreatment with a single dose of 12 mg/kg i.v., all six ADCs reduced thetumour volume and improved survival compared to the antibody trastuzumabitself and control vehicle, without negatively affecting body weight. Itwas concluded that conjugates that contain a relatively short linkerhave a better (antitumor) efficacy than the corresponding conjugate witha relatively long linker, and that both the nature of the linker and thenature of the drug were demonstrated to have an effect on efficacy aswell.

Breast cancer remains the most common malignancy among women worldwide.Breast cancer is a heterogeneous disease, which exhibits a wide range ofclinical behaviours and prognoses. Breast cancer is an abnormalmalignant growth of epithelial cells of the milk lobules or ducts of themammary gland. The cancer tissue can be exclusively located on the placeof origin (cancer in situ) or can have invaded through the basementmembrane into the surrounding tissue (invasive cancer). Metastaticcancer occurs as soon as the cancer cells have spread by way of lymphand blood vessels to other organs. Histological differentiation andcharacterization of the breast cancer cells is performed with use ofbiomarkers.

Molecular classification of breast cancer for therapeutic decisionsmainly consists of the assessment of the estrogen receptor (ER),progesterone receptor (PR), and human epidermal growth factor receptor 2(HER2) expression status. This implies that globally three types ofbreast cancer can be discerned: (1) breast cancer tissue with expressionof hormone receptor (ER or PR) without over-expression of HER2, (2)breast cancer tissue with over-expression of HER2, with or withoutexpression of hormone receptor (HR), and (3) breast cancer tissue thathas no therapeutically relevant hormone receptor or HER2 receptorexpression, so-called triple negative breast cancer (TNBC).

Breast cancer patients with hormone receptor (HR) positive cancer tissuestatus (ca. 60-70% of all breast cancer patients) have a betterprognosis than those without or with minimal hormone receptor status. Onthe contrary, patients whose tumour has an IHC 3+ or IHC 2+/FISH(fluorescence in situ hybridization) positive status (occurring in about20% of all breast cancer cases) have a worse prognosis in comparisonwith breast cancer patients whose tumour has a lower grade of HER2membrane expression or a FISH negative amplification rate. Now thatpatients with hormone receptor positive and HER2 over-expressing breastcancer tissue have the option of targeting therapy, triple negativebreast cancer implies the worst prognosis, as only chemotherapy withlimited efficacy is available for these patients whose tumour is ER, PRand HER2 negative.

Herceptin™ (trastuzumab), a recombinant humanized IgG1 monoclonalantibody against HER2, was approved in the US by the FDA in 1998 foradjuvant treatment of HER2 over-expressing breast cancer and for thetreatment of metastatic HER2 over-expressing breast cancer and gastriccancer, i.e. HER2 IHC 3+ or HER2 IHC 2+/FISH positive. The drug wasapproved in Europe by the EMA in 2000.

Clinical studies with patients who have metastatic breast cancer diseasehave demonstrated, that there is only clinical relevant efficacy oftrastuzumab treatment if the patient has a tumour with HER2 IHCover-expression or FISH positive gene amplification. For this reason,current HER2 testing algorithms are aimed at identifying those patientsmost likely to achieve a significant benefit from HER2 targeting.Whereas HER2 membrane expression is biologically a continuum from low tohigh over-expression, approved IHC tests, like the HercepTest™ (Dako,Glostrup, Denmark), categorize HER2 status on a semi-quantitative scaleranging from 0 to 3+. An IHC score of 3+is assigned if there is a strongcircumferential membrane staining in >10% of the cancer cells. FISHpositive gene amplification is assigned if the amplification raterelative to the centromere is ≥2.0. It identifies patients who mighthave a benefit of treatment with trastuzumab or other HER2 targetingagents. A review of 6,556 breast cancers revealed that about 92% oftumours with a HER2 score of 3+ had FISH positive gene amplification.Conversely, HER2 amplification was observed at lower rates in tumourswith scores of 2+ (23.3%), 1+ (7.4%), and 0 (4.1%). With HER2amplification as an established predictor of response to HER2 targetingagents, the current algorithm calls for FISH testing of tumours with aHER2 IHC score of 2+.

Ado-trastuzumab emtansine or trastuzumab emtansine (Kadcyla™, T-DM1) isan ADC in which trastuzumab is conjugated to the cytotoxic maytansineanti-tubulin agent DM1. T-DM1 has antitumor activity in tumour xenograftmodels that are not responding to therapy with trastuzumab as singleagent. In the Phase 3 EMILIA trial, patients with HER2 positive advancedbreast cancer, previously treated with trastuzumab and a taxane, wererandomly assigned to receive T-DMI or lapatinib plus capecitabine. T-DM1treatment effectuated significantly longer progression-free and overallsurvival time in comparison to the treatment of the control group.

Kadcyla™ (T-DM1) was approved in the US by the FDA in February 2013 forthe treatment of patients with HER2-positive metastatic breast cancerwho received prior treatment with trastuzumab and a taxane. The drug wasapproved in Japan by the MHLW (Ministry of Health, Labour and Welfare)in September 2013 and in Europe by the EMA in November 2013. Thecurrently approved regimen comprises a dosage of 3,6 mg/kg body weighti.v. every three weeks. A dosage of 2.4 mg/kg body weight i.v. weekly isinvestigated in an ongoing Phase II study with a combination of T-DM1and capecitabine for the 2^(nd) line treatment of patients with breastcancer or gastric cancer and in an ongoing Phase Ill study toinvestigate T-DM1 against a taxane as 2^(nd) line treatment of patientswith gastric cancer. A Phase ITT study is also ongoing for thecombination of T-DM1 with pertuzumab for the treatment of patients withHER2 positive, locally advanced, or metastatic breast cancer.

Despite the improvement that the introduction of T-DM1 in clinicalpractice brought over trastuzumab for the treatment of HER2-positivemetastatic breast cancer, the use of T-DM1 is associated with a numberof serious side-effects, most importantly thrombocytopenia,hepatotoxicity, and neuropathy (irreversible axonal degeneration).Furthermore, neither trastuzumab nor T-DM1 are authorized for thetreatment of human solid tumours and haematological malignancies withmoderate or low HER2 expression, i.e. IHC 2+ or 1+ and/or FISH negativeHER2 amplification status of the cancer tissue.

In analogy to breast cancer, HER2 expression indicates a poor prognosisfor patients with ovarian cancer (A. Berchuck et al., 1990, Cancer Res.,50, 4087-4091; H. Meden and W. Kuhn, 1997, Eur, J. Obstet. & Gynecol.Reprod. Biol., 71, 173-179). SKOV3 cells are derived from the ascitesfluid of a patient with ovarian adenocarcinoma. This cell line isover-expressing HER2 and is frequently used for in vitro and in vivoexplorative investigation of HER2 targeting agents. Trastuzumab andpertuzumab have several anti-cancer effects in this cell line (N.Gaborit et al., 2011, J. Biol. Chem., 286, 13, 11337-11345). Monotherapywith the anti-HER2 antibodies trastuzumab and pertuzumab thus far hadmodest efficacy (G. M. Mantia-Smaldone et al., 2011, Cancer ManagementRes. 3, 25-38; S. P. Langdon et al., 2010, Expert Opin. Biol. Ther.10:7, 1113-1120), The antitumor effect is markedly increased if a HER2targeting antibody is combined with chemotherapy (S. Makhija et al.,2010, J. Clin. Oncol., 28:7, 1215-1223; I. Ray-Coquard et al., 2008,Clin. Ovarian Cancer, 1:1, 54-59).

Further, a high medical need exists for the treatment of late stagebladder cancer disease. Chemotherapy, e.g. the combination of cisplatinand gemcitabine for advanced or metastatic bladder cancer, has limitedefficacy as it effectuates in the mean a response rate under 50%,whereas patients have an overall survival time of 6 to 12 months. Incase of resistance to chemotherapy there is no standard therapy optionat all. HER2 positivity was significantly associated with reducedcomplete response rates (50% versus 81%, p=0.026) after chemo-radiation(A. Chakravarti et al., 2005, Mt. J. Radiation Oncology Biol. Phys.,62:2, 309-317). Addition of trastuzumab to a regimen of paclitaxel andcarboplatin as first line therapy of HER2 positive advanced bladdercancer showed an overall response rate of 70% and an overall survivaltime of 14.1 months in a Phase II study (M. H. A. Hussain et al., 2007,J. Clin. Oncol., 25:16, 2218-24). In a casuistic application, a patientwith a tumor relapse after standard chemotherapy responded to thecombination of trastuzumab, paclitaxel and carboplatin (D.Arnsellem-Ouazana et al., 2004, Ann. Oncol., 15, 3, 538).

In case of invasive non-small-cell lung cancer adenocarcinoma, HER2mutation and amplification are related with unfavorable outcome (M.Suzuki et al., 2014, Lung Cancer,http://dx.doi.org/10.1016/j.lungca.2014.10.014). In lung cancer patientswith HER2 mutation, a disease control rate of 93% could be effectuatedwith trastuzumab-based therapies (J. Mazieres et al., 2013, J. Clin.Oncol., 31:16, 1997-2004). Chemo-resistance of lung cancer often isassociated with enhanced HER2 expression (C.-M. Tsai et al., 1993, J.Natl. Cancer Inst., 85:11, 897-901; Z. Calikusu et al., 2009, J. Exp.Clin. Cancer Res., 28:97) and resistance to tyrosine kinase inhibitorsis correlated with enhanced HER2 amplification (K. Takezawa et al.,2012, Cancer Discov. 2(10), 922-33).

Patients with early or advanced prostate cancer mostly receive anandrogen receptor targeting therapy. There is a cross-talk in thesignaling functions of the androgen receptor and HER2 (F.-N. Hsu et al.,2011, Am. J. Physiol. Endocrinol. Metab., 300:E902-E908; L. Chen et al.,2011, Clin. Cancer Res., 17(19), 6218-28). HER2 activation suppressesthe expression of the androgen receptor (C. Cai et al., 2009, CancerRes., 69(12), 5202-5209), increased HER2 expression is associated withPSA progression, rapid proliferation and poor prognosis (S. Minner etal., 2010, Clin. Cancer Res., 16(5), 1553-60; S. F. Shariat et al.,2007, Clin. Cancer Res., 13(18), 5377-84). Increased expression of HER2seems to be involved in progression to androgen independence in about aquarter of prostate cancer cases (J. M. S. Bartlett et al., 2005, J.Pathol., 205, 522-529).

Pancreatic cancer is among the most lethal human solid tumors due to itsinsidious onset and resistance to therapy. Gemcitabine or thecombination of 5-FU, leucovorin, irinotecan, and oxaliplatin can helpprolong life in patients with advanced disease (H. Burris and A. M.Storniolo, 1997, Eur. J. Cancer 33(1):S18-S22; T. Conroy et al., 2011,N. Engl. J. Med. 364(19):1817-25). More recently, it was reported thatHER2 expression is also prevalent in pancreatic cancer with an equalproportion of 10% designated as HER2 2+ and 3+. Based on this fact,HER2-targeted treatment comprising trastuzumab is considered as a viableoption in this patient population based on effects observed inpre-clinical models [C. Larbouret et al., 2012, Neoplasia 14(2),121-130).

Using accepted staining and scoring methods, over-expression of HER2 wasobserved in approximately 6% of colorectal cancer (CRC) patients (A. N.Seo et al., 2014, PLoS ONE, 9(5): e98528). Based on this, HER2-targetingtreatment may be effective in this subset of CRC patients. Two clinicaltrials have investigated the benefit of trastuzumab-containingcombination therapy in advanced or metastatic CRC and clinical responseswere observed in these trials providing evidence of treatment efficacy(R. K. Ramanathan et al., 2004, Cancer Invest. 22(6): 858-865; J. Clarket al., 2003, Proc. Am. Soc. Clin. Oncol. 22: abstr 3584). Moreover, onestudy suggested the inclusion of trastuzumab therapy as part oftreatment regimens for (anti-EGFR monoclonal antibody)cetuximab-resistant CRC patients (A. Bertotti et al., 2011, CancerDiscov. 1(6): 508-523).

The management of advanced head and neck squamous cell cancer orcarcinoma (HNSCC) consists of multiple-modality therapy with surgery,radiation, and chemotherapy. Beckhardt et al. reported high HER2over-expression in 16% of cell line samples, and moderate and low HER2expression in 31% and 35% of samples, respectively (R. N. Beckhardt etal., 1995, Arch. Otolaryngol. Head Neck Surg. 121:1265-1270). Thisillustrates the potential therapeutic potential of trastuzumab treatmentin HNSCC.

In 1999, Gorlick et al. reported over-expression of HER2 in 20 of 47osteosarcoma samples, and showed that these patients had a poor responseto therapy and a decreased rate of survival compared with patients whosetumors did not over-express this antigen (R. Gorlick et al., 1999, J.Clin. Oncol. 17:2781-8). Hence, HER2 emerged as a promising candidatefor targeted biologic therapy in this indication. Recent findings fromclinical investigation using trastuzumab indicate that anti-HER2treatment can be safely delivered in combination withanthracycline-based chemotherapy and dexrazoxane (D. Ebb et al., 2012,J. Clin. Oncol. 30(20), 2545-2551).

Further, HER2 over-expression is seen in approximately one-third ofacute lymphoblastic leukaemia (ALL) patients, even more frequent in thepresence of the Philadelphia translocation. Inhibition of HER2 inducesapoptosis of the leukemia cells in vitro (M. E. Irwin et al., 2013, PLoSONE, 8:8, e70608). In a Phase II study, it was demonstrated thattrastuzumab treatment of refractory or relapsing adult B-ALL patientswith HER2 over-expression in malignant B-cells resulted in an overallresponse rate of 13%, which shows the response of this disease to a HER2targeting agent (P. Chevalier et al., Blood, 2012, DOI10.1182/blood-2011-11-390781).

Hence, there is a need for new HER2-targeted therapies, notably fortreating patients with tumours and malignancies that have (i) a moderateor low IHC status, and/or (ii) a negative FISH status, and/or (iii) ahormone receptor (HR) negative status of the cancer tissue.Particularly, new regulatory approved therapies are needed for thetargeted treatment of triple negative breast cancer (TNBC).

BRIEF DESCRIPTION OF THE PRESENT INVENTION

The present invention relates to duocan-nycin-containing antibody-drugconjugates (ADCs) for use in the treatment of human solid tumours andhaematological malignancies expressing HER2, in particular breastcancer, gastric cancer, bladder cancer, ovarian cancer, lung cancer,prostate cancer, pancreatic cancer, colorectal cancer, head and necksquamous cell cancer, osteosarcoma, and acute lymphoblastic leukaemia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Antitumor activity of SYD985 compared to T-DM1 in MAXF-1162 PDXmodel (breast cancer, adenocarcinoma, HER2 IHC 3+, HER2 FISH positive)(CRO: Oncotest).

FIG. 2. Antitumor activity of SYD985 compared to T-DM1 in HBCx-34 PDXmodel (breast cancer, ductal carcinoma, HER2 IHC 2+, HER2 FISH negative,ER and PR positive) (CRO: XenTech).

FIG. 3. Antitumor activity of SYD985 compared to T-DM1 in MAXF 119 PDXmodel (breast cancer, invasive ductal carcinoma, HER2 IHC 1+, HER2 FISHnegative, ER and PR negative, i.e. triple negative breast cancer) (CRO:Oncotest).

FIG. 4. Antitumor activity of SYD985 compared to T-DMI in HBCx-10 PDXmodel (breast cancer, ductal adenocarcinoma, HER2 IHC 1+, HER2 FISHnegative, ER and PR negative, i.e. triple negative breast cancer) (CRO:XenTech).

FIG. 5. Antitumor activity of SYD985 compared to T-DM1 in MAXF-MX1 PDXmodel (breast cancer, invasive ductal carcinoma, HER2 IHC 1+, HER2 FISHnegative, ER and PR negative, i.e. triple negative breast cancer) (CRO:Oncotest).

FIG. 6. Antitumor activity of SYD985 compared to T-DM1 in ST313 PDXmodel (breast cancer, HER2 IHC 2+, HER2 FISH negative, ER and PRpositive) (CRO: Start).

FIG. 7. Antitumor activity of SYD985 compared to T-DMI in GXA3057 PDXmodel (gastric cancer, HER2 IHC 1+, HER2 FISH negative) (CRO: Oncotest).

FIG. 8. Antitumor activity of SYD985 compared to T-DMI in GXA3067 PDXmodel (gastric cancer, HER2 IHC 2+, HER2 FISH positive) (CRO: Oncotest).

FIG. 9. Antitumor activity of SYD985 compared to T-DM1 in GXA3054 PDXmodel (gastric cancer, HER2 IHC 3+, HER2 FISH positive) (CRO: Oncotest).

FIG. 10. Antitumor activity of SYD985 compared to T-DMI in GXA3038 PDXmodel (gastric cancer, HER2 IHC 2₊, HER2 FISH negative) (CRO: Oncotest).

FIG. 11. Antitumor activity of SYD985 in BXF439 PDX model (bladdercancer, HER2 IHC 3+, HER2 FISH positive) (CRO: Oncotest).

FIG. 12. Antitumor activity of SYD983 in SKOV3 cell line-derivedxenograft model (ovarian cancer, HER2 IHC 2+, HER2 FISH positive) (CRO:Piedmont).

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention relates to duocarmycin-containing ADCs for use inthe treatment of human solid tumours and haematological malignanciesexpressing HER2.

In one embodiment, the present invention provides a compound of formula(I)

wherein

anti-HER2 Ab is an anti-HER2 antibody or antibody fragment,

n is 0-3, preferably 0-1,

m represents an average DAR (drug-to-antibody ratio) of from 1 to 4,

R¹ is selected from

y is 1-16, and

R² is selected from

for use in the treatment of human solid tumours and haematologicalmalignancies expressing HER2, in particular for use in the treatment ofhuman solid tumours.

In another embodiment, the present invention relates to a compound offormula (I), wherein anti-HER2 Ab is an anti-HER2 antibody or antibodyfragment, n is 0-1, m represents an average DAR of from 1 to 4,preferably from 2 to 3, R¹ is selected from

y is 1-16, preferably 1-4, and R² is selected from

In a further embodiment, the present invention relates to a compound offormula (I), wherein the anti-HER2 Ab is an anti-HER2 monoclonalantibody, n is 0-1, m represents an average DAR of from 2 to 3,preferably from 2.5 to 2.9, R¹ is selected from

y is 1-4, and R² is selected from

In yet another embodiment, the present invention relates to a compoundof formula (I), wherein the anti-HER2 Ab is trastuzumab or a biosimilarthereof, n is 0-1, m represents an average DAR of from 2 to 3,preferably 2.5 to 2.9, R¹ is selected from

y is 1-4, and R² is selected from

In a preferred embodiment, the present invention relates to a compoundof formula (II), comprising trastuzumab or a biosimilar thereof

The compound of formula (II) that is referred to as SYD985 in thepresent specification has an average DAR of from 2.6 to 2.9. SYD983 offormula (II) has an average DAR of 10.

In the structural formulae shown in the present specification, nrepresent an integer from 0 to 3, while m represents an averagedrug-to-antibody ratio (DAR) of from 1 to 4. As is well-known in theart, the DAR and drug load distribution can be determined, for example,by using hydrophobic interaction chromatography (HIC) or reversed phasehigh-performance liquid chromatography (RP-HPLC). HIC is particularlysuitable for determining the average DAR.

Examples of human solid tumours which can be treated in accordance withthe present invention are breast cancer, gastric cancer, bladder cancer,ovarian cancer, lung cancer, prostate cancer, pancreatic cancer,colorectal cancer, head and neck squamous cell cancer, and osteosarcoma,particularly breast cancer, gastric cancer, bladder cancer, ovariancancer, lung cancer, and prostate cancer, more particularly breastcancer, gastric cancer, and bladder cancer (see also S. Scholl et al.,2001, Ann. Oncol., 12(1): S81-S87). An example of a haematologicalmalignancy which can be treated in accordance with the present inventionis acute lymphoblastic leukaemia (ALL). The scope of the presentinvention however is not restricted to these specific examples.

In one embodiment, the present invention provides a compound of formula(I) or (II) for use in the treatment of breast cancer, gastric cancer orbladder cancer, particularly breast cancer or gastric cancer, especiallybreast cancer. Said breast cancer either is hormone receptor (ER and/orPR) positive or negative, advantageously ER and PR negative.

In another embodiment, the present invention provides a compound offormula (I) or (II) for use in the treatment of human solid tumoursshowing moderate or low expression of HER2 (i.e. HER2 IHC 2+ or 1+).

In yet another embodiment, the present invention provides a compound offormula (I) or (II) for use in the treatment of human solid tumourswithout HER2 gene amplification (i.e. HER2 FISH negative).

Unexpectedly, the present inventors have found that theduocarmycin-containing ADC compounds of the present inventionparticularly can be used for the treatment of human solid tumours,especially breast cancer and gastric cancer, with a moderate or lowexpression of HER2 (i.e. HER2 IHC 2+ or 1+) and/or without HER2 geneamplification (i.e. HER2 FISH negative). Neither trastuzumab nor T-DM1obtained marketing approval for the treatment of patients having suchtumours. Furthermore, as shown in the Examples and Figures herein below,T-DM1 lacks efficacy in such tumours. Hence, the duocarmycin-containingADC compounds of the present invention can be used for the treatment ofpatient groups for which there is no current HER2-targeted therapyavailable. The duocarmycin-containing ADC compounds that were tested inmice bearing an N87 (i.e. HER2 IHC 3+ gastric tumour) xenograft inExample 15 of WO2011/133039A indeed showed efficacy after a single i.v.dose of 12 mg/kg. However, there is nothing in this document suggestingthe person skilled in the art to test—let alone expect to findefficacy—in lower grade HER2-expressing tumours (i.e. HER2 IHC 2+ or 1+)and/or without HER2 gene amplification (i.e. HER2 FISH negative),already at a dose of 3 mg/kg.

The present inventors surprisingly further found that theduocarmycin-containing ADC compounds of formula (I) or (II) show animproved in vivo antitumor activity in animal tumour models as comparedto T-DM1 (see Examples and Figures) and trastuzumab when administered atthe same dose. Notably, it was found that the improvement was thehighest in tumour models with the lowest grade of HER2 expression (i.e.IHC HER2 1+), in particular in (triple negative) breast cancer andgastric cancer.

In an advantageous embodiment of the present invention the human solidtumour is breast cancer or gastric cancer showing moderate or low HER2expression (i.e. HER2 IHC 2+ or 1+) without HER2 gene amplification(i.e. HER2 FISH negative).

In a particularly advantageous embodiment of the present invention, thehuman solid tumour is triple negative breast cancer (i.e. HER2 IHC 2+or1+, HER2 FISH negative, and ER and PR negative).

Typically, the antitumor activity is evaluated first in (human) tumourcell lines in vitro followed by evaluation in vivo. The antitumoractivity of the ADCs falling within the scope of the present inventionadvantageously is evaluated in animal models, typically immunodeficientmice bearing a subcutaneous xenograft. The xenograft can either be a(human) tumour cell line or a patient-derived (primary) tumour.Preferably, the animal model is a patient-derived tumour xenograft (PDX)model.

Human tumours in PDX models retain the biological characteristics of theoriginal tumour as assessed by microscopic examination. PDX models areroutinely used now in many academic institutions and are offeredcommercially by a number of Contract Research Organizations (CROs)including Jackson Lab (USA), Oncotest (Germany), Molecular Response(USA), Charles River (USA), Oncodesign (France), XenTech (France),Champions Oncology (USA), and Start (USA). Many have shown the retentionof characteristic morphologic and immunohistochemical features of theoriginal human tumour in the xenograft. Besides the close relationshipwith regards to biological characteristics, PDX models have a very goodpredictive value for therapeutic clinical outcome. In general, one couldstate that reports from different sources indicate at least 90% correctreplication of the response to therapy in the PDX compared to that inthe patient, both in terms of sensitivity and resistance of the tumourto therapy (Website Champions Oncology,http://www.championsoncology.com/translational-oncology-solutions/predictive-value;Fiebig et al., 1984, Behring Inst. Mitt. 74:343-352; Hidalgo et al.,2011, Mol. Cancer Ther. 10:1311-1316).

In accordance with the present invention, the anti-HER2 antibody orantibody fragment can be any antibody or antibody fragment able to bindHER2, e.g. an IgG1 antibody having the complementary determining regions(CDRs) of trastuzumab or an antibody that shows competitive binding withtrastuzumab. A preferred antibody is a monoclonal anti-HER2 antibody. Aparticularly preferred monoclonal antibody is trastuzumab or abiosimilar thereof.

Antibody-drug conjugate (ADC) compounds of formula (I) and (II) inaccordance with the present invention have the linker-drug conjugated tothe antibody through the S-atom of a cysteine residue, i.e. they arecysteine-linked antibody-drug conjugates. The cysteine residue caneither be a natural cysteine residue which is present in the heavyand/or light chain of the antibody (Ab) and forms inter-chain disulfidebonds, or an engineered cysteine residue which is introduced into the Abat one or more suitable positions in the heavy and/or light chain. Thepresent invention is particularly drawn to ADC compounds wherein thelinker-drug is conjugated through inter-chain disulfide bonds of Abs,more particularly monoclonal Abs (mAbs). Antibodies of differentantibody classes contain different numbers of interchain disulfidebonds. For example, IgG1 antibodies typically have four inter-chaindisulfide bonds, all four located in the hinge region, and after(partial) reduction of the disulfide bonds the linker-drug is randomlyattached to free thiol groups.

Compounds of formula (I) and (II) for use in accordance with the presentinvention can be obtained according to methods and procedures that arewell known to a person skilled in the art. Conjugation throughinter-chain disulfide bonds can occur after complete or partialreduction of said disulfide bonds. Suitable methods for preparing suchcompounds can be found in the description and examples of Applicant'sWO2011/133039A. In particular, Example 15 of WO2011/133039A describesthe partial reduction of trastuzumab to generate 2 free thiol groups permAb and conjugation with a number of linker-drugs to ADCs having anaverage DAR of approx. 2. It is easily understood by those skilled inthe art how to obtain ADCs having an average DAR of from 1 to 4.Examples 7 and 8 of W02005/084390A describe partial reduction, partialreduction/partial re-oxidation, and complete reduction strategies for(partial) loading of antibodies with the linker-drug vcMMAE.

IHC and FISH status of the tumour tissue are determined using knowntests, procedures and equipment. In accordance with the presentinvention HER2 gene amplication can be measured using eitherfluorescence (FISH) or chromogenic (CISH) or any other in situhybridization test. Suitable tests for determination of the HER2membrane expression status of the tumour tissue like the HercepTest™(Dako Denmark) are commercially available. Further HER2 IHC tests aremarketed by Ventana Medical Systems (PATHWAY anti-HER2/neu), BiogenexLaboratories (InSite™ HER2/neu), and Leica Biosystems (Bond Oracle™ HER2IHC). FISH/CISH tests can be obtained from Abbott Molecular (PathVysionHER2 DNA Probe Kit), Life Technologies (SPOT-Light® HER2 CISH Kit), DakoDenmark (HER2 CISH PharmDx™ Kit), Dako Denmark (HER2 FISH PharmDx™ Kit),and Ventana Medical Systems (INFORM HER2 Dual ISH DNA Probe Cocktail).FISH positive means a FISH amplification ratio ≥2.0 (e.g. by using DakoHER2 FISH PharmDX™ test kit). FISH negative means a FISH amplificationratio <2.0.

HER2 expressing tumours which can be advantageously treated inaccordance with the present invention are breast cancer and gastriccancer, particularly breast cancer, most particularly triple negativebreast cancer. Unexpectedly, the present inventors have found that theADC compounds in accordance with the present invention notably wereeffective in breast cancer PDX models which are HER2 IHC 2+ or 1+andFISH negative, in triple negative breast cancer PDX models, and ingastric cancer PDX models which are HER2 IHC 2+ or 1+ and FISH negative,as shown in the Examples and Figures herein below. In view of the factthat PDX models have a very good predictive value for therapeuticclinical outcome, these findings particularly offer a new HER2-targetedtreatment option for breast and gastric cancers for which there iscurrently no such approved treatment option available.

The present invention also relates to the use of a compound of formula(I) or (II) for the treatment of patients having human solid tumours orhaematological malignancies expressing HER2, in particular of humansolid tumours which are HER2 IHC 2+or 1+and/or which are HER2 FISHnegative as described herein above.

The present invention further relates to the use of a combination of acompound of formula (I) or (II) with a therapeutic antibody and/or achemotherapeutic agent, for the treatment of human solid tumours andhaematological malignancies expressing HER2, in particular human solidtumours, most particularly for the treatment of triple negative breastcancer.

In one embodiment of the present invention, the therapeutic antibody ispertuzumab, bevacizumab, ramucirumab or trastuzumab and thechemotherapeutic agent is i) a taxane, particularly docetaxel,paclitaxel, nab-paclitaxel, or cabazitaxel, ii) a mitotic inhibitor,particularly eribulin, vinorelbine or vinblastine, iii) a DNA damagingagent, particularly 5-fluoro-uracil, capecitabine, gemcitabine,temozolomide, cisplatin, carboplatin, oxaliplain, cyclophosphamide orifosfamide, iv) an anti-folate, particularly pemetrexed or methotrexate,v) an anthracycline, particularly mitoxantrone, doxorubicin, liposomaldoxorubicin, epirubicin, daunorubicin or valrubicin, more particularlydoxorubicin, vi) an mTOR (mammalian target of rapamycin) inhibitor,particularly temsirolimus or everolimus, vii) a topo-isomeraseinhibitor, particularly irinotecan or topotecan, viii) a tyrosine kinaseinhibitor, particularly gefitinib, erlotinib, pazopanib, crizotinib,lapatinib or afatinib, ix) an androgen receptor modulating agent,particularly enzalutamide or abiraterone acetate, x) a steroid hormone,particularly prednisone, xi) a hormonal therapeutic agent, particularlytamoxifen, xii) an aromatase inhibiting or steroid modifying agent,particularly anastrozole, letrozole, fulvestrant or exemestane, or xiii)a PARP inhibitor, particularly olaparib. The person skilled in the artwill have no difficulty in selecting suitable combination therapies foruse in the treatment of human solid tumours and haematologicalmalignancies expressing HER2.

In another embodiment of the present invention, the therapeutic antibodyis pertuzumab and the chemotherapeutic agent is a taxane, particularlydocetaxel or paclitaxel, or an anthracycline, particularly doxorubicin,epirubicin, daunorubicin or valrubicin, more particularly doxorubicin.

The present invention further relates to the use of a combination of acompound of formula (I) or (II) with another ADC, such as for exampleT-DM1, for the treatment of human solid tumours and haematologicalmalignancies expressing HER2, in particular human solid tumoursexpressing HER2.

The present invention further relates to a pharmaceutical compositioncomprising a compound of formula (I) or (II) or a combination with atherapeutic antibody and/or a chemotherapeutic agent thereof asdescribed herein above, and one or more pharmaceutically acceptableexcipients.

Typical pharmaceutical formulations of therapeutic proteins such asmonoclonal antibodies and (monoclonal) antibody-drug conjugates take theform of lyophilized powders or cakes, which require (aqueous)dissolution (i.e. reconstitution) before intravenous infusion, or frozen(aqueous) solutions, which require thawing before use. Particularly, inaccordance with the present invention the pharmaceutical composition isprovided in the form of a lyophilized cake.

Suitable pharmaceutically acceptable excipients for inclusion into thepharmaceutical composition (before freeze-drying) in accordance with thepresent invention include buffer solutions (e.g. citrate, histidine orsuccinate containing salts in water), lyo protectants (e.g. sucrose,trehalose), tonicity modifiers (e.g. sodium chloride), surfactants (e.g.polysorbate), and bulking agents (e.g. mannitol, glycine). Excipientsused for freeze-dried protein formulations are selected for theirability to prevent protein denaturation during the freeze-drying processas well as during storage.

The sterile, lyophilized powder multi-dose formulation of Herceptin™contains 440 mg trastuzumab, 400 mg α,α-trehalose dihydrate, 9.9 mgL-histidine.HCl, 6.4 mg L-histidine, and 1.8 mg polysorbate 20, USP.Reconstitution with 20 ml of Bacteriostatic or Sterile Water forInjection (BWFI or SWFI) yields a multi-dose solution containing 21mg/nil trastuzumab at a pH of approximately 6. The sterile, lyophilizedpowder single-use formulation of Kadcyla™ contains upon reconstitution20 mg/ml ado-trastuzumab emtansine, 0.02% w/v polysorbate 20, 10 mMsodium succinate, and 6% w/v sucrose with a pH of 5.0.

A therapeutically effective amount of the compound of formula (I) or(II) for use in accordance with the present invention lies in the rangeof about 0.01 to about 15 mg/kg body weight, particularly in the rangeof about 0.1 to about 10 mg/kg, more particularly in the range of about0.3 to about 10 mg/kg body weight. This latter range corresponds roughlyto a flat dose in the range of 20 to 800 mg of the ADC compound. Thecompound of the present invention is administered weekly, bi-weekly,three-weekly or monthly, for example weekly for the first 12 weeks andthen every three weeks until disease progression. Alternative treatmentregimens may be used depending upon the severity of the disease, the ageof the patient, the compound being administered, and such other factorsas would be considered appropriate by the treating physician.

EXAMPLES PDX HER2 Gene Amplification Testing

Amplification of the HER2 gene was determined by in situ hybridization(ISH) in formalin-fixed, paraffin-embedded human breast cancer tissuespecimens using FDA approved tests from Ventana Medical Systems (INFORMHER2 Dual ISH DNA Probe Cocktail) or Abbott Molecular (PathVysion HER2DNA Probe Kit). Protocols used were as detailed by the suppliers of thetests.

PDX HER2 IHC Staining

Tissue sections of formalin-fixed, paraffin-embedded tumour xenograftsamples were prepared. HER2 was bound by using a suitable Ab, forinstance a polyclonal rabbit anti-human HER2 (DAKO Cat# A0485) antibodyand detected by a suitable secondary Ab, for instance biotinylated goatanti-rabbit IgG (JacksonImmuno research, Cat# 111-065-04) and a Biozol(Cat# VEC-PK-4000) ABC kit. Staining was evaluated semi-quantitativelyon a suitable microscope, for instance using a Zeiss Axiovert 35microscope. Staining was interpreted as immunoreactivity, based on thenumber of stained tumour cells as well as the completeness and intensityof the membrane staining.

0: <10% of the tumour cells exhibit membranous stain.

1: >10% of the tumour cells exhibit membranous stain, but incompletestain of surface.

2: >10% of the tumour cells exhibit weak or moderate membranous staindistributed all over the surface.

3: >30% of the tumour cells exhibit strong membranous stain distributedall over the surface.

Known HER2 positive (IHC 3+) and HER2 negative (IHC 0) control tumourslides were included in every HER2 staining procedure.

PDX and Cell Line-Derived Xenograft Animal Studies

All animal studies were approved by local animal ethical committees andwere performed according to local ethical guidelines of animalexperimentation. Female immunodeficient nu/nu mice (4-6 weeks of age) orSOD mice from a professional animal breeder like Harlan or Charles Riverwere used and randomization was performed according to the detailedprotocols of the respective CROs, as described for instance by Fiebig etal. in Cancer Genomics & Proteomics 4: 197-210, 1997.

All breast and gastric PDX studies were performed testing SYD985head-to-head to T-DM1, since the latter ADC was approved for treatmentof patients with HER2-positive metastatic breast cancer and approval forT-DM1 in HER2-positive gastric cancer is currently being pursued.Studies in other indications (bladder and ovarian) were done usingSYD985 only, since T-DM1 is not an approved drug in those indications.Mice were treated with either vehicle, 3 mg/kg SYD985 or 3 mg/kg T-DM1in all breast PDX models (FIGS. 1-6) and with vehicle, 10 mg/kg SYD985or 10 mg/kg T-DM1 in all gastric PDX models (FIGS. 7-10). Mice weretreated with vehicle or 10 mg/kg SYD985 in the bladder PDX model (FIG.11) and with vehicle or 15 mg/kg SYD983 in the cell line-derived ovarianxenograft model (FIG. 12). All treatments were conducted at day 0 by asingle dose, i.v. injection into the tail vein. Data, depicted as meantumour volume±S.D., consists of 6-8 animals per experimental group. Bodyweight and tumour size were measured twice weekly. The tumour volume wasdetermined by a two-dimensional measurement with callipers. Terminationcriteria included among others a tumour volume >2000 mm³ or a bodyweight loss >30%. Tumour size of individual animals was processed usingGraphPad Prism. The results are shown in FIGS. 1 to 12.

First-In-Human Clinical Study

A two-part first-in-human phase I study (with expanded cohorts) with theantibody-drug conjugate SYD985 (trastuzumab vc-seco-DUBA) is beingperformed to evaluate the safety, pharmacokinetics and efficacy inpatients with locally advanced or metastatic solid tumours (i.e.NCT02277717). Part I is the dose-escalation part in which a low dose ofSYD985 is given to three cancer patients (females or males having solidtumours of any origin). If it is well tolerated, a higher dose of SYD985will be given to three other cancer patients. This will continue untilit is not safe anymore to increase the dose further. In part II of thestudy, several groups of patients with a specific type of cancer(including breast and gastric tumours) will receive the SYD985 doseselected for further development. All patients from both parts of thestudy (it is estimated that a total of 76 patients will be enrolled)will receive SYD985 (intravenous) infusions every three weeks untilprogression of the cancer or unacceptable toxicity develops.

1-15. (canceled)
 16. A method of treating cancer in a human patient,comprising administering to the patient a therapeutically effectiveamount of a compound of formula (I)

wherein anti-HER2 Ab is an anti-HER2 antibody or antibody fragment, n is0, 1, 2, or 3, m represents an average DAR of from 1 to 4, R¹ isselected from the group consisting of

y is 1-16, and R² is selected from the group consisting of

wherein the patient has a solid tumor expressing HER2 and the solidtumor is HER2 IHC 2+ or 1+, and wherein the solid tumor is breastcancer, gastric cancer, bladder cancer, ovarian cancer, lung cancer,prostate cancer, pancreatic cancer, colorectal cancer, head and necksquamous cell cancer, or osteosarcoma.
 17. The method of claim 16,wherein anti-HER2 Ab is an anti-HER2 antibody or antibody fragment, n is0 or 1, m represents an average DAR of from 1 to 4, R¹ is selected fromthe group consisting of

y is 1-16, and R² is


18. The method of claim 17, wherein anti-HER2 Ab is an anti-HER2monoclonal antibody, n is 0 or 1, m represents an average DAR of from 2to 3, R¹ is

y is 1, 2, 3, or 4, and R² is


19. The method of claim 18, wherein the anti-HER2 Ab is trastuzumab. 20.The method of claim 16, wherein the compound of formula (I) is acompound of formula (II)


21. The method of claim 20, wherein the compound of formula (II) has anaverage DAR of from 2.5 to 2.9.
 22. The method of claim 21, wherein thecompound of formula (II) has an average DAR of from 2.6 to 2.9.
 23. Themethod of claim 20, wherein the compound of formula (II) is administeredin an amount of from about 0.3 mg/kg to about 10 mg/kg.
 24. The methodof claim 16, wherein the solid tumor is breast cancer, gastric cancer,bladder cancer, ovarian cancer, lung cancer, or prostate cancer.
 25. Themethod of claim 24, wherein the solid tumor is breast cancer or gastriccancer.
 26. The method of claim 20, wherein the solid tumor is HER2 FISHnegative.
 27. The method of claim 26, wherein the solid tumor is breastcancer.
 28. The method of claim 16, further comprising administering aneffective amount of a therapeutic antibody or a chemotherapeutic agent,or a combination thereof.
 29. The method of claim 28, wherein the cancertreated is breast cancer.
 30. The method of claim 28, wherein thetherapeutic antibody is pertuzumab and the chemotherapeutic agent is ataxane or an anthracycline.
 31. The method of claim 30, wherein thetaxane is docetaxel or paclitaxel, and the anthracycline is doxorubicin,epirubicin, daunorubicin or valrubicin.
 32. A method of treating bladdercancer in a human patient, comprising administering to the patient atherapeutically effective amount of a compound of formula (I)

wherein anti-HER2 Ab is an anti-HER2 antibody or antibody fragment, n is0, 1, 2, or 3, m represents an average DAR of from 1 to 4, R¹ isselected from the group consisting of

y is 1-16, and R² is selected from the group consisting of


33. The method of claim 32, wherein anti-HER2 Ab is an anti-HER2antibody or antibody fragment, n is 0 or 1, m represents an average DARof from 1 to 4, R¹ is selected from the group consisting of

y is 1-16, and R² is


34. The method of claim 33, wherein anti-HER2 Ab is an anti-HER2monoclonal antibody, n is 0 or 1, m represents an average DAR of from 2to 3, R¹ is

y is 1, 2, 3, or 4, and R² is


35. The method of claim 34, wherein the anti-HER2 Ab is trastuzumab. 36.The method of claim 32, wherein the compound of formula (I) is acompound of formula (II)


37. The method of claim 36, wherein the compound of formula (II) has anaverage DAR of from 2.5 to 2.9.
 38. The method of claim 37, wherein thecompound of formula (II) has an average DAR of from 2.6 to 2.9.
 39. Themethod of claim 32, wherein the compound of formula (II) is administeredin an amount of from about 0.3 mg/kg to about 10 mg/kg.
 40. The methodof claim 32, further comprising administering an effective amount of atherapeutic antibody or a chemotherapeutic agent, or a combinationthereof
 41. The method of claim 40, wherein the therapeutic antibody ispertuzumab and the chemotherapeutic agent is a taxane or ananthracycline.